Compositions containing decitabine, 5azacytidine and tetrahydrouridine and uses thereof

ABSTRACT

Compositions comprising decitabine and tetrahydrouridine or 5-azacytidine and tetrahydrouridine for the treatment of cancer are disclosed. The compositions in the form of a pill is administered to a human subject sequentially or alternately guided by the measurement of predictive biomarkers.

RELATED APPLICATIONS DATA

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. application Ser. No. 15/780,796 filed Jun. 1, 2018, now U.S. Pat.No. 11,376,270 issued Jul. 5, 2022, which is a 35 U.S.C. § 371 NationalPhase Entry Application of International Application No. PCT/US16/64935filed Dec. 5, 2016, which designates the U.S. and which claims thebenefit under 35 U.S.C. § 119(e) from U.S. Provisional Application Ser.No. 62/262,839 filed Dec. 3, 2015 and Ser. No. 62/429,292 filed Dec. 2,2016.

FIELD OF THE INVENTION

This application relates generally to compositions containing decitabine(DEC), 5-azacytidine (5AZA) and tetrahydrouridine (THU) and the methodsof using the compositions for the treatment of cancers and blooddisorders in a subject. Specifically, the compositions in the form of apill include DEC and THU or 5AZA and THU or a combination of DEC, 5AZAand THU as a non-cytotoxic treatment for cancer or other blood disordersin a subject.

BACKGROUND OF THE INVENTION

Radiation and drug treatments for cancer intend to engage physiologicpathways that can terminate growth and division of cells. The specificphysiologic pathway that almost all cancer drugs are attempting toengage is the p53/p16-mediated apoptosis pathway (cytotoxicity). Sincep53 and p16 are the two most commonly inactivated genes in cancer, thisp53/p16-dependence of cancer drugs and radiation is a fundamental basisfor treatment resistance and relapse. Hence there is a need for noveltreatment approaches that do not use the p53/p16 system to terminate thecell growth and division of cancer cells.

Pre-clinical and clinical studies have shown that non-cytotoxicdepletion of the enzyme DNA methyl transferase 1 (DNMT1) by decitabine(DEC) and 5-azacytidine (5AZA) could be an effective form of treatmentthat does not require or rely on the p53/p16 system (Negrotto S., et al.2011 Cancer Res 71(4):1431-1441). Yet 5AZA or DEC by themselves are notcurative, neither in pre-clinical in vivo models of cancer nor in theclinic. It would therefore be desirable to utilize mechanisms of cancerresistance to p53-independent treatment with 5AZA or DEC.

SUMMARY OF THE INVENTION

The present disclosure has identified that cancer resistance top53-independent treatment with 5AZA or DEC can be effectively overcomeby logical, non-toxic and clinically applicable solutions. Specifically,the present disclosure provides compositions of non-cytotoxicTHU-DEC/THU-5AZA and methods for overcoming cancer resistance byadministration of compositions comprising non-cytotoxic levels ofTHU-DEC and administering the composition in an alternating regimeni.e., alternating the intake of the composition that may be in the formof a pill or tablet containing THU-DEC and THU-5AZA for defined timeperiods.

Accordingly, in one embodiment an oral composition for inducing safe,non-cytotoxic (p53/p16-independent) differentiation-mediated cell cycleexits of cancer cells is provided.

The composition for oral administration comprising a nucleoside analogdrug that can bind irreversibly and deplete the enzyme DNA methyltransferase 1 (DNMT1), a potent cytidine deaminase (CDA) inhibitor andone or more pharmaceutically acceptable excipients, wherein thenucleoside analog drug and the potent cytidine deaminase inhibitor arepresent in therapeutically effective amounts sufficient to inducedifferentiation of a refractory and resistant cancer cell withoutcytotoxicity to normal tissues. The nucleoside analog drug that depletethe DNMT1 enzyme present in the composition is DEC. The nucleosideanalog drug in one embodiment may also be 5AZA. The CDA inhibitor in thecomposition may be tetrahydrouridine (THU) or certain tetrahydrouridinederivatives such as 2′-fluorinated tetrahydrouridine derivatives. Inthis embodiment when the nucleoside analog drug that deplete DNMT1 isDEC it may be present in amounts ranging from 1-10 mg/m². When thenucleoside analog drug present in the oral composition is 5AZA it may bepresent in amounts ranging from 10-100 mg/m². The CDA inhibitor THU inthe oral composition may be present in amounts ranging from about 400mg/m² to about 500 mg/m².

A treatment composition for inducing non cytotoxic differentiation mayexist in the form of a pill or tablet or capsule. The treatmentcomposition in one embodiment may comprise the nucleoside analog drugDEC and THU and one or more pharmaceutically acceptable carriers. Thetreatment composition in another embodiment may comprise 5AZA, THU andone or more pharmaceutically acceptable carriers. The treatmentcomposition in some embodiment may comprise a therapeutically effectiveamount of both of the nucleoside analog drugs (DEC or 5AZA) combinedwith THU in a single pill.

The treatment composition may be administered to a subject aftermeasurement of a predictive biomarker, including but not limited to DCKin the cancer cells. Development of predictive biomarkers may assist atreating physician to select the right treatment regimen in order to bemore effective in treating an especially refractory and aggressivemetastatic cancer in a subject. In this embodiment the predictivebiomarker may be the pyrimidine metabolism enzyme deoxycytidine kinase(DCK) of cancer cells. The DCK enzyme is known to phosphorylate and trapDEC in cells and several cancer cells avoid the DEC-induced DNMT-1depletion by resorting to an alternative pyrimidine metabolism enzymeUCK2, which however renders the cancerous cells vulnerable to the drug5-5AZA. The measurement of DCK of cancer cells may be performed beforeor during or after administration of the oral composition by functionalmolecular imaging with—positron emission tomography (PET) utilizing aDCK-dependent probe or by QRT-PCR.

In a treatment method for inducing non cytotoxic differentiation ofcancer cells in any mammalian tissue, a first treatment compositioncomprising DEC, THU and one or more pharmaceutically acceptable carriersand a second treatment composition comprising 5-5AZA, THU and one ormore pharmaceutically acceptable carriers may each be administered to apatient in need thereof sequentially or alternately or simultaneously.In this embodiment the tetrahydrouridine may also include derivativessuch as the 2′-fluorinated tetrahydrouridine derivatives. The first andthe second compositions may be administered after, or during or beforemeasuring the predictive biomarkers in cancer cells.

In one embodiment the first and the second treatment compositions mayeach be in the form of a pill or they may be combined within a singlepill. When the compositions of the present disclosure exist in the formof a pill the THU or the THU derivative may specifically be located onthe surface of the pill, while the DEC or 5AZA may be located inside thepill. Such differential location of the nucleoside analog drugs, DEC or5AZA and the THU on a pill may ensure that the THU or the THU derivativemay be bioavailable about 1 minute to 180 minutes or 15 minutes to about180 minutes before either DEC or 5AZA and more preferably between 30 toabout 60 minutes before either DEC or 5AZA. In another embodiment theDEC or 5AZA may be coated or embedded in a polymer to facilitate adelayed release of the drugs compared to the release of a polymer coatedor embedded THU or THU derivative.

Upon oral administration of the treatment composition the combination ofDEC and THU may produce at least about 10-fold improvement in the oralbioavailability, low C_(max) and multi-hour T_(max) needed fornon-cytotoxic DNMT-1 depletion by DEC or 5AZA. Because of knownoff-target or toxic effects of DEC and 5AZA, optimizing DEC or 5AZA todeplete DNMT1 in vivo may have powerful therapeutic benefits.Accordingly, the treatment composition may result in a peak plasma DEClevel of about 0.05 μm to about 0.5 μm in a human subject, or a peakplasma 5AZA level of about 0.5 to about 5 μm. In this embodiment atreating physician may prescribe a pill containing DEC and THU (or)-5AZA and THU in a sequential or intermittent fashion in such a way thatthe patient stricken with cancer takes a pill containing DEC and THUfirst followed by days or weeks apart by a pill containing 5AZA and THUor vice versa.

In one embodiment a method for treating any tumor, metastatic tumor orcombination thereof in a tissue of a subject or blood malignancies orblood disorders in a subject is provided. The method comprising thesteps of (i) administering a therapeutically effective amount of a firstcomposition in the form of a pill comprising a first nucleoside analogdrug, a cytidine deaminase inhibitor and a pharmaceutically acceptablecarrier, (ii) administering a second composition in the form of a pillcomprising a second nucleoside analog drug, a cytidine deaminaseinhibitor and a pharmaceutically acceptable carrier, wherein thenucleoside analog drug in the first composition may be DEC, thenucleoside analog drug in the second composition may be 5AZA and thecytidine deaminase inhibitor in the said first composition and the saidsecond composition is THU or THU derivative such as 2′-fluorinatedtetrahydrouridine derivatives. In this embodiment the first and thesecond compositions may be administered sequentially or alternately orsimultaneously. Additionally, the first and second compositions may beadministered before or during or after measuring the level of predictivebiomarker in the cancer cells of a subject.

A treatment selection involves the determination of the DCK level in apatient requiring treatment and prescribing the appropriate nucleosideanalog drug in combination with THU (i.e., DEC and THU or 5AZA and THUor both) in such a way to steer the cancerous tissue towards adifferentiation pathway. The drug compositions in the form of a pill maybe prescribed once a week or once every two weeks or between once tothree times per week to patients stricken with cancer or bloodmalignancies or disorders. In yet other embodiment, the composition inthe form of a pill may be prescribed between once every two weeks to asoften as three times per week in patients at risk of developinghematological or solid malignancy, or at risk of having a relapse in aprevious diagnosis of hematological or solid malignancy or hematologicalor blood disorders.

In one embodiment a kit for treating a tumor or refractory tumor orhematological tumor or blood disorders is provided. The kit maycomprise: (i) a pill comprising DEC, THU or THU derivative and acarrier, (ii) a pill comprising 5AZA, THU or THU derivative and acarrier and (ii) an instruction manual. In another embodiment the kitmay comprise (i) a capsule comprising a mixture of DEC/THU microspheresor microparticles and 5AZA/THU microspheres or microparticles and (ii)an instruction Manual. The pill or capsule may be formulated in such away that THU or THU derivative is always bioavailable at least 1 minuteto about 60 minutes before the nucleoside analog drugs, DEC or 5AZA. Inone embodiment the THU or THU derivative may be bioavailable from 1minute to about 15 minutes or 15 minutes to 30 minutes, 30 minutes to 45minutes, 45 minutes to 60 minutes before DEC or 5AZA.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of CDKN2A and TP53deletion/mutations that are prognostic in Renal Cell Carcinoma (RCC).

FIG. 2 is a graphical representation of the genes, VHL, PBRM1 and Pax8,amplified or deleted in RCC.

FIG. 3 is a graphical representation showing the hypermethylated WT1promoter CpG in RCC compared to non-paired non-cancerous kidney.

FIG. 4 is a graphical representation of the high expression ofdifferentiation drivers and paradoxically epigenetic repression of keyepithelial differentiation target genes (e.g., WT1 and GATA3) in RCC.

FIG. 5 is a comprehensive documentation of CoActivator and CORepressorInteractions of Pax8 by LC/MSMS.

FIG. 6 is graphical representation of the PAX8 interactions towardsCoActivators and CoRepressors due to DNMT1 depletion by non-cytotoxicconcentrations of DEC.

FIG. 7 is a graphical representation of the DCK and UCK2 expressionlevels that correlate with DEC and 5AZA sensitivity respectively.

FIG. 8 is a graphical representation at relapse of the MDS/AML cellsfrom patients treated with Dec having lower DCK and higher UCK2 levelsand vice-versa in 5-Aza treated patients.

FIG. 9 . is a graphical representation of the efficacy of THU-DEC 3×/wk.alternating with THU-55AZA 3×/wk. in xenotransplant models ofpatient-derived AML

FIG. 10 is a flow chart showing how the biomarkers can guide logicalmeasures to extend the response to drugs and salvage relapse.

DETAILED DESCRIPTION

The present disclosure relates to compositions that contain a nucleosideanalog drug in combination with a potent cytidine deaminase inhibitorand pharmaceutically acceptable excipient. The nucleoside analog drugpresent in the compositions can be any known nucleoside deoxycytidineanalog drug that are also DNA demethylating agents including but notlimited to DEC, 5AZA, gemcitabine, etc.

The term “nucleoside analogues” refer to any nucleoside that contain anucleic acid analog and a sugar which upon phosphorylation act as ametabolite inhibitor. Specifically the nucleoside analog in thisdisclosure refers to cytidine analogs including but not limited to DECwith unmodified sugar and 5AZA. DEC (5-5-aza-2′-deoxycytidine) mimics anatural component of DNA and is relatively unique amongst the largefamily of nucleoside analogue drugs in that it can irreversibly bind toand deplete the enzyme DNA methyl transferase 1 (DNMT1), and also thatthe unmodified sugar does not terminate DNA chain elongation, enablingdepletion of DNMT1 without cytotoxicity. In cancer cells, DNMT1depletion by the drug DEC prevents the repression of differentiationgenes and renews the differentiation of the cancer cells—the abnormalgrowth of the cancer cells is caused by a block in their normaldifferentiation process, which is relieved by DEC. Of special note,DNMT1 depletion in normal stem cells increases their self-renewal; thatis, DNMT1 depletion increases the number of normal stem cells—theopposite of its effects on cancer cells. Therefore, DNMT1 depletion byDEC could be an effective and very safe, well-tolerated cancer therapy.

In one embodiment of the invention, the pharmacologic objective of anon-cytotoxic therapy, defined as treatment that does not inducecytotoxicity (apoptosis) of normal dividing cells, is to maximize thetime-above-threshold concentration of DEC or 5AZA for depleting DNMT1(≥0.05-0.2 μM for DEC, ≥0.5-2 μM for 5AZA) in vivo, while avoiding highpeak levels (≥0.5-1 μM for DEC, ≥5-10 μM for 5AZA) that damage DNA. Thislow C_(max) but extended T_(max) exposure will also be produced in solidtissues, into which there is minimal even negligible distribution ofcurrent formulations of DEC and 5AZA, because of high CDA expression insolid tissues including the liver, kidneys, intestines and brain. TheDNMT1-depleting effect of DEC or 5AZA in the present disclosure iscontemplated to be intermittent because continuous exposure to DEC or5AZA may cause accumulation of DEC or 5AZA in normal cells to levelsthat induce cytotoxicity. For example the currently known route ofadministration, regimens, and formulations of DEC or 5AZA can producehigh peak levels of the drug that are known to kill normal cells insensitive tissues such as the bone marrow through cytotoxicity butproduce very brief time-above-threshold concentrations for depletingDNMT1 in the bone marrow but especially in solid tissues which highlyexpress CDA. Also, the currently known route of administration,regimens, and formulations of DEC or 5AZA do not increase and distributeexposure times to increase depletion of DNMT1 while avoiding thecontinuous or sustained exposures that result in accumulation of DEC or5AZA in normal bone marrow cells to levels that cause cytotoxicity.Instead, these current approaches mimic traditional regimens withpulse-cycled administrations intended for cytotoxic treatment intents.Importantly, the destruction of DEC and 5AZA by the enzyme cytidinedeaminase (CDA) produces an abbreviated half-life in vivo of <20 minutes(despite an in vitro half-life of 5-9 hours) (Liu, Z., et al. 2006 RapidCommon Mass Spectrum 20:1117-1126); and this drastic reduction inhalf-life is a significant barrier to effective in vivo translation ofin vitro observations, especially for solid tissue cancers which residein tissues which intrinsically express high levels of CDA. High peaklevels of DEC or 5AZA in the plasma and bone marrow also result frompharmacogenomic variation in CDA (Gilbert, J. A., et al. 2006 Clin.Cancer Res 12, 1794-1803; Kirch, H. C., et al. 1998 Exp. Hematol. 26,421-425; Yue, L., et al. 2003 Pharmacogenetics 13, 29-38) that produceslarge inter-individual variation in pharmacokinetics (PK) and clinicaleffects. Also, malignant cells can develop resistance by destroying DECwith CDA (Ohta, T., et al. 2004 Oncol Rep 12:1115-1120; Hubeek, I., etal. 2005 Br J Cancer 93:1388-1394; Huang, Y., et al. 2004 Cancer Res64:4294-4301) and the malignant cells may find sanctuary from DECtherapeutic effects by residing in tissues with high levels of CDA(Ebrahem, Q., 2012 Oncotarget 3(10):1137-45).

To address these multiple fundamental pharmacologic limitations of DECand 5AZA, an oral route of administration of DEC or 5AZA in combinationwith the CDA inhibitor THU is preferred herein. Such oral administrationof DEC or 5AZA in combination with THU substantially decreases peaklevels compared to current parenteral administration routes whilesubstantially increasing the time-above-threshold concentrations fordepleting DNMT1; the oral administration enables chronic, frequent butnot daily (i.e., metronomic) therapy to sustain life-long therapeuticeffects, while allowing cell division and avoiding cytotoxicity; theoral combination administration enables distribution of DEC or 5AZA intosolid tissues which are sites of cancers and eliminates cancer cellsanctuary in CDA-rich organs such as the liver. In brief; thecombination enables treatment of solid tissue cancers. Moreover, theoral administration facilitates wide-spread use of the drugs across theglobe. Cytidine deaminase (CDA) is an enzyme that is highly expressed inthe liver and intestine and rapidly destroys DEC and 5AZA within thebody. In humans, the CDA gene is subject to non-synonymous singlenucleotide polymorphisms which produce variants of cytidine deaminasethat have differences in enzymatic activity of 3-fold or more (Gilbert,J. A., et al. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C., et al.1998 Exp Hematol 26, 421-425; Yue, L., et al. 2003 Pharmacogenetics 13,29-38).

THU is known to inhibit the enzyme Cytidine deaminase or CDA and hasmany desirable characteristics including but not limited to—exhibiting abenign toxicity profile, well-characterized PK, ability to prevail overthe intestinal and liver first-pass barriers to oral bioavailability ofDEC and 5AZA, producing more predictable effects of a DEC or 5AZA dosefrom one individual to other, increasing the time-above thresholdconcentration of DEC or 5AZA for depleting DNMT1, removing the sanctuarysites for malignant cells from DEC or 5AZA therapeutic effects, etc.Hence, in one embodiment the invention provides a composition for oraladministration wherein the composition comprises about DEC from about1-10 mg/m², or 5AZA in amounts ranging from 10-100 mg/m², together withthe CDA inhibitor THU in amounts ranging from about 400 mg/m² to about500 mg/m². Although the CDA inhibitor used in the composition of thepresent disclosure is THU, a person skilled in the art may appreciatethe fact that other CDA inhibitors such as zebularine may be usedinstead of THU. In some other embodiment the CDA inhibitor used in thetreatment composition may be a commonly known THU derivative such as2′-fluorinated tetrahydrouridine derivatives synthesized as inhibitorsof CDA and described in Ferraris et al. 2014 J Med Chem. 57(6):2582-88or U.S. Pat. No. 8,329,665 or EP2447272.

Since malignant cells can develop resistance to combination of DEC-THUby shifting pyrimidine metabolism from salvage by DCK to salvage byUCK2, or develop resistance to combination 5AZA-THU by shiftingpyrimidine metabolism from salvage with UCK2 to salvage with DCK, bothdrugs are contemplated to be used alternately or sequentially, tologically address their respective mechanisms of resistance. In anotherembodiment administration of the composition results in a plasmaconcentration of DEC of about 0.05 to about 0.5 μM or a concentration of5AZA of about 0.5 to about 5 μM. In yet another embodiment, acomposition is administered once a week or once every two weeks topatients suffering from metastatic cancer. In still another embodiment,the composition is administered between once to three times per week topatients with cancer. In some embodiment, a composition of the inventionis administered between once every two weeks to as often as three timesper week in patients at risk of developing hematological or solidmalignancy, or at risk of having a relapse in a previous diagnosis ofhematological or solid malignancy.

5AZA incorporates to a larger extent into RNA than DNA, theincorporation of which results in the inhibition of the proteinproduction. When it incorporates into DNA it covalently binds with DNAmethyltransferases that prevent DNA synthesis and subsequentcytotoxicity. In a preferred embodiment the DEC and 5AZA used in thecomposition for oral administration is a free drug form that are free ofsalts.

In one embodiment, the disclosure provides a composition for oraladministration in the form of a pill, comprising THU and DEC or THU and-5AZA, wherein the THU or ‘THU derivative (2’-fluorinatedtetrahydrouridine derivatives) is released more quickly than the DEC or5AZA. It will be appreciated by one skilled in the art that suchdifferential and/or dual release of the drugs of the present disclosuremay be effectuated by any known means in the art. The ‘differential ordual release’ refers to the release at different time points of ‘a drug’in a pill containing more than one drug, and/or bioavailability of thedrugs at different time points after ingestion. In a differentialrelease formulation for example, the THU might be subject to a fasterdissolution rate, or the THU might be located at the surface of thepill, while the DEC or 5AZA is located inside the pill. Because ofeither the location of THU or the coating or embedding of THU in apolymer or suitable vehicle the THU or THU derivative is bio-availableabout 1 minute to about 180 minutes before the DEC or 5AZA, in another,about 15 to about 60 minutes before the DEC or 5AZA, in another, about30 or 60 minutes before the DEC or 5AZA. The THU may also beadministered separately, in succession with THU first, then DEC or 5AZA.The term “bio-available”, as referred to herein, refers to when theactive agent (THU or DEC or 5AZA) can be absorbed and used by the body.“Orally bio-available” indicates that the agent has been taken by mouthand can be absorbed and used by the body.

In another embodiment the composition for oral administration may be asingle pill (is also used herein as ‘capsule or tablet’) that comprisesboth nucleoside analog drugs, DEC and 5AZA in addition to THU or THUderivative and at least one pharmaceutically acceptable excipient. Inthis embodiment the DEC or 5AZA and THU are differentially located onthe said pill so that THU or THU derivative is bioavailable at leastabout 1 minute to about 180 minutes before DEC or 5AZA. In thisembodiment the drugs, DEC and 5AZA may each exist as either coated orembedded microspheres or microparticles that facilitate the release ofthe drugs after the THU or THU derivative. This composition comprisingboth cytidine analog drugs is administered to patient at defined timeperiods such as between once to three times per week to patients withaggressive and refractory cancer. In yet another embodiment, thiscomposition is administered between once every two weeks to as often asthree times per week in patients at risk of developing hematological orsolid malignancy, or at risk of having a relapse in a previous diagnosisof hematological or solid malignancy.

The term “pharmaceutically acceptable excipient”, as used in thecomposition refers to carriers and vehicles that are compatible with theactive ingredient (for example, a compound of the composition) of apharmaceutical composition of the invention (and preferably capable ofstabilizing it) and not deleterious to the subject to be treated. Forexample, solubilizing agents that form specific, more soluble complexeswith the compounds of the invention can be utilized as pharmaceuticalexcipients for delivery of the compounds. Suitable carriers and vehiclesare known to those of extraordinary skill in the art. The term“excipient” as used herein will encompass all such carriers, adjuvants,diluents, solvents, or other inactive additives. Suitablepharmaceutically acceptable excipients include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical compositions of theinvention can also be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like, which do notdeleteriously react with the active compounds of the invention.

In another embodiment, the composition of the invention is stored with adesiccant. This could serve to extend the shelf-life of a composition ofthe invention and facilitate its distribution and use on a global scale.

The term “treating”, as used herein, refers to altering the diseasecourse of the subject being treated. Therapeutic effects of treatmentinclude, without limitation, preventing occurrence or recurrence ofdisease, alleviation of symptom(s), diminishment of direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

Pharmaceutical Compositions

The pharmaceutical compositions of this disclosure can be prepared asdescribed in U.S. application Ser. No. 13/141,669 (issued as U.S. Pat.No. 9,259,469) and Ser. No. 13/414,546 (issued as U.S. Pat. No.9,265,785), incorporated herein in its entirety. In an embodiment,pharmaceutical compositions and non-toxic dosage forms of the inventioncomprise about 0.1 to about 0.5 mg/kg CDEC or 1 to about 5 mg/kg of 5AZAand about 10 to about 50 mg/kg THU and a pharmaceutically acceptableexcipient, in relative amounts and formulated in such a way that a givenpharmaceutical composition or dosage form releases the repression ofdifferentiation pathways in the tumor cell or decreases the aberrantrepression of differentiation-related genes in the tumor cell. Thetherapeutically effective amount of the pharmaceutical compositions anddosage forms of the invention may represent or result in one or more ofthe following: (i) peak levels of DEC or 5AZA in the plasma of patientsthat are below thresholds above which there is damage to DNA in normaldividing cells sufficient to cause apoptosis (cytotoxicity), (ii)exposure times sufficient to deplete DNMT1 in cancer cells that areextended to hours from minutes (5 to 20-fold extension of the Tmax) (DECor 5AZA alone have plasma exposure times of minutes) but are nonethelessbelow thresholds that cause intra-cellular levels of DEC or 5AZA toaccumulate to levels that induce damage to DNA in normal dividing cellssufficient to cause apoptosis (cytotoxicity). In short, exposure timeoptimized for non-cytotoxic depletion of DNMT1 (iii) a 5 to 20-foldincrease in the oral bioavailability of DEC or 5AZA, (iv) Distributionof DEC or 5AZA into CDA-rich tissues in which cancer cells otherwisefind sanctuary from DEC or 5AZA treatment effects. This is the sameprinciple underlying oral bioavailability, that is, the ability of DECor 5AZA to now distribute through the intestines and liver, (v) induceexpression of genes and proteins necessary for cell cycle exits bydifferentiation (p53/p16-independent cell cycle exits) by cancer cells,and (v) Non-cytotoxic mechanism of action, that spares normal dividingcells. Avoidance of toxicity in this way facilitates frequentintermittent treatment to increase the fraction of the cancer exposed toDNMT1-depleting effects.

For example, in some embodiment, the pharmaceutical compositions anddosage forms of the invention comprise about 1 to about 5 mg/kg 5AZA or0.1 to about 0.5 mg/kg of DEC and about 10 to about 50 mg/kg THU and apharmaceutically acceptable excipient, in relative amounts andformulated in such a way that a given pharmaceutical composition ordosage form releases the repression of differentiation pathways in thetumor cell or decrease the aberrant repression ofdifferentiation-related genes in the tumor cell.

The pharmaceutical composition or the dosage forms may also includeadditional active agent including chemotherapeutic agents known in theart. The pharmaceutical compositions may also be administered alone orin combination with other known compositions for treating cancer in amammalian subject. The term “in combination” may include simultaneous orsequential administration of the composition of this disclosure. Thesequential administration may include administration of the disclosedtreatment composition followed by any known composition or vice-versa.

The administration of the compositions may be carried out in single ormultiple doses. In general, the therapeutically-effective compounds ofthis disclosure are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight. In some embodiments asubject requiring treatment of tumor may receive a tablet or capsulecontaining 10 mg/kg of THU and 0.2 mg/kg of DEC or 2 mg/kg of 5AZA twiceweekly on consecutive days or once a week or once in 3 days for about aweek or two weeks.

To enable differential release of THU or THU derivative and thenucleoside analog drugs, a sustained release composition wherein theactive component is derivatized with differentially degradable coatingse.g., by microencapsulation, multiple coatings may be formulated. Insome embodiments nanoparticles of the pharmaceutical composition can beprepared as described in U.S. Published application Ser. No. 14/046,343,which is incorporated here in its entirety. These compositions ensurethat THU is bioavailable about 1 minute to about 180 minutes before DECand/or -5AZA. In another embodiment the micro or Nano formulationsensure that the THU is bioavailable about 15 to about 60 minutes or 30to about 60 minutes before DEC and/or -5AZA.

The compositions of the present disclosure may also additionally includeother agents that reduce the rate by which the active compounds willdecompose. Such agents may include stabilizers including but not limitedto antioxidants, ascorbic acid, pH buffers or salt buffers.

Predictive Biomarkers

Developing “predictive or companion” biomarkers to guide treatmentselection is a powerful and effective tool in selecting a meaningfulindividualized treatment regimen such as the dosage and schedule for anysubject in need of treatment. Accordingly a predictive biomarker in theform of an enzyme profile is measured during the treatment regimen toassist the treating physician determine the appropriate treatmentregimen for the subject. DNA synthesis and renewal of genetic materialare essential for cell proliferation, and high expression of thepyrimidine salvage enzyme DCK is a pan-cancer hall mark ofproliferation. Accordingly, radio-labeled nucleoside analogs may beemployed for imaging tumor proliferation through the salvage pathway ofDNA synthesis. 1-(2′-deoxy-2′-[¹⁸F]-fluoro-β-D-arabinofuranosyl)cytosine(TFAC), which like DEC, is a deoxycytidine analogue, is one suchradio-tracer. Since active DEC needs to be activated by DCK to produceits DNMT1-depleting effect, cancer DCK expression reliably predictssensitivity to DEC, across the full spectrum of cancer histology andgenetics. Accordingly, TFAC-Positron emission tomography (PET) may beemployed to measure DCK-dependent cancer cell proliferation.

In some embodiment the predictive bioassay may be conducted before,during and after the treatment with the disclosed compositions. Theassay may be conducted to select the appropriate treatment regimen. Forexample if the assay before or during treatment indicates a high FACuptake which correlate with the response to oral THU-DEC, the treatingphysician may continue to prescribe a pill or tablet that comprisesTHU-DEC. If the FAC uptake is low or decreasing in the context ofincreasing tumor size indicating the dominance of cancer cells that usethe alternative pyrimidine metabolism enzyme, UCK2—a mechanism ofresistance to DEC, then the physician may prescribe the tablet orcapsule comprising THU-5AZA.

Critically, these measurements do not just provide scientific insightinto mechanisms of resistance, but guide application of rationalclinical measures to overcome resistance and extend response: (i)failure to achieve molecular-PD in the context of no side-effects ofthis non-cytotoxic therapy can guide rational increases in the dosage orfrequency of oral THU-DEC (FIG. 9 ). (ii) Resistance with re-increasingvariant allele frequency burden concurrent with loss of molecular-PD andshift in pyrimidine metabolism enzymes can guide rational switch intherapy to oral THU-5AZA (FIG. 9 ). Optimal application will likelyincorporate oral THU-DEC administered for one alternating with oralTHU-55AZA administered the next week on a chronic, long-term basis(FIGS. 7-9 ).

Methods of Treatment

In one embodiment a pill comprising the treatment composition of thepresent disclosure may be administered to a cancer patient in need oftreatment. The cancer may be a solid or liquid cancer or may be anaggressive and refractory cancer of any known tissues in a subject. Thetreatment composition of the present disclosure may also be effective intreating hematological malignancies or blood disorders such as but notlimited to sickle cell disease, hemoglobinopathies, thalassemias or anyassociated conditions that are elaborated in U.S. patent applicationSer. No. 13/414,546, now issued as U.S. Pat. No. 9,265,785, incorporatedherein in its entirety. ‘Treatment or treating’ refers to any knownphysical and/or biochemical and/or molecular changes in the cancertissue. For example treatment may refer to amelioration of a tumor sizeor a known cancer biomarker protein such as the level of DNMT1 in normaland malignant cells or may be molecular, as in de-repression of specificdifferentiation genes or all of the above. The method of treatmentincludes assaying a predictive biomarker before, during or after thetreatment. The predictive biomarker may inform the treating physicianthe appropriate dosage and schedule to prescribe to the patient. Forexample patients may receive the drugs of the present disclosure asfollows: oral THU 10 mg/kg, followed by oral DEC 0.2 mg/kg 60 minutesafter the THU, twice weekly on consecutive days. The treatment mayinclude administering a pill comprising THU-DEC or THU-5AZA or a pillcontaining both DEC and 5-5AZA, wherein the THU or THU derivative isbioavailable about 1 minute to about 180 minutes or 15 to about 180minutes before DEC and/or 5AZA.

The treatment method may include the administration of a firstcomposition comprising DEC, THU or a THU derivative and at least onepharmaceutically acceptable excipient followed by days or weeks apart byalternate administration of a second composition comprising 5AZA, THU ora THU derivative and at least one pharmaceutically acceptable excipient.This sequential or alternate treatment method may be followed forseveral weeks to several months.

In some aspects the method of treatment includes an additionaltherapeutic modality including but not limited to radiation therapy or acytotoxic chemotherapy agent such as an anti-metabolite, doxorubicin,HDAC inhibitors, anti-viral agents, anti-retroviral agent or anycombination of these agents as described in detail in U.S. Publishedapplication Ser. No. 14/046,343, incorporated here in its entirety.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1

A Need for Treatments Mechanistically Distinct from Current Options

Historically, most drugs evaluated in RCC attempted to terminateproliferation using the p53/p16 apoptosis system (cytotoxicity). Fordecades, such treatments proved to be toxic and ineffective because RCCcells, like most cancer cells, are apoptosis-resistant relative tonormal cells, through genetic abnormalities in key p53/p16-system genessuch as CDKN2A. The first drugs approved by the FDA to treat RCCinhibited signaling pathways (e.g., AKT/mTOR) that stabilize MYC proteinthat is the master coordinator of cell growth and division. Thus, thesedrugs impede RCC growth directly through effects on MYC protein and alsoindirectly, by inhibiting angiogenesis needed to support RCC growth.Although such therapies are a clear advance, durable response is rare(median survival 18-30 months), and p53/p16 alterations still impactresponse, robust to multi-variate analyses (FIG. 1 ). Moreover,responses in 2^(nd) or 3^(rd) line are substantially less durable thanresponses in 1^(st) line, suggesting some overlap in mechanisms ofresistance to approved drugs. It is thus indicated and appropriate toevaluate mechanistically distinct treatments that explicitly usepathways other than p53/p16 to terminate RCC cell growth and division.

PBRM1 Inactivation in RCC and Epithelial Differentiation Therapy

An unexpected revelation from RCC genomic studies is essentiallyuniversal inactivating alterations of genes for coactivator subunits,especially PBRM1, but also others (SETD2, SMARCC1, SMARCA2, ARID1A)(FIG. 2 ). These observations are recent, and the pathway function ofthese coregulators in normal kidney cells, or the pathways by which lossof function conferred clonal advantage, were unknown. We conjecturedthat a possibly connected observation was epigenetic suppression of keyepithelial terminal differentiation genes (e.g., GATA3, WT1) in RCC(FIGS. 3, 4 ). This epigenetic repression of late-differentiation geneswas particularly intriguing because RCC cells express un-mutated mastertranscription factors that drive epithelial differentiation (e.g., PAX8,PAX2) at relatively preserved mRNA and high protein levels (FIG. 4 ).Another pertinent observation was correlation of increased chromatinrepression marks with worse prognosis in multiple RCC studies. In thelast funding cycle we connected these observations experimentally,discovering that PBRM1/BAF mediates transcription activation by PAX8, amaster driver of mesenchymal-epithelial transition11-16 that is highlyexpressed in RCC17-22. PBRM1 loss causes unbalanced recruitment to PAX8of corepressor counterparts to PBRM1/BAF (e.g., DNMT1), repressinginstead of activating proliferation-terminatingepithelial-differentiation target genes (FIG. 5 ). Importantly,inhibiting these specific corepressors (e.g., DNMT1) restored expressionof multiple PAX8 target genes, renewing epithelial differentiation andphysiologic, p53/p16-independent cell cycle exits (FIG. 6 ).

Example 2

A Need for Mechanism-Based Predictive Biomarkers

DNA synthesis and renewal of genetic material are essential for cellproliferation, and high expression of the pyrimidine salvage enzyme DCKis a pan-cancer hall mark of proliferation. Accordingly, radio-labelednucleoside analogs have been developed for imaging tumor proliferationthrough the salvage pathway of DNA synthesis.1-(2′-deoxy-2′-[18F]-fluoro-β-D-arabinofuranosyl)cytosine (FAC), likeDec a deoxycytidine analogue, is one such radio-tracer. It has beendocumented that similar to Dec, however, FAC did not adequatelydistribute into solid tissues such as the liver and kidneys, because ofrapid deamination by CDA. It was then found that THU-FAC (TFAC)co-injection solves this problem, producing high FAC uptake inhepatocellular cancer against the background liver in a woodchuck animalmodel of viral infection induced primary hepatocellular cancer. As shownin FIG. 10 the use of predictive biomarker assay will help determinewhether the frequency or dose of the THU-Dec should be increased.

In this way, TFAC-PET could suggest rational salvage therapy. Ifvalidated, it could also have a role in upfront treatment selectionbetween THU-Dec versus THU-5Aza. Additionally, it could be that TFAC-PETdoes capture an aspect of RCC biology that correlates well with responseand outcomes to other RCC drugs (sunitinib etc.). In other words, it isconceivable that TFAC-PET could fulfill the broad predictive biomarkerrole that FDG-PET has not been able to meet. Potential utilitydemonstrated here can justify evaluation for such a broader purpose.

Example 3

Treatment Resistant Malignant Cells in Patients Treated with DECUpregulate UCK2 and Downregulate DCK and Vice-Versa in Patients Treatedwith 5AZA

Bone marrow aspirate specimens were obtained from patients before and atthe time of relapse while on treatment with DEC or 5AZA, and expressionof the pyrimidine metabolism enzymes DCK and UCK2 was measured byquantitative real-time PCR. As shown in FIG. 8 during relapse MDS/AMLcells from patients treated with Dec have lower DCK and higher UCK2, andvice versa in 5Aza treated patients.

Example 4

Non-Cytotoxic Treatment Using THU-DEC 3×/Wk. Alternating with THU-5AZA3×/Wk. Strikingly Extends Response Compared to DEC Alone, or THU-DECAlone, in Xenotransplant Models of Patient-Derived AML

Primary AML cells (1×10⁶ cells) from the bone marrow of a patient withAML were xenotransplanted into NSG mice. Five days after theinoculation, mice were treated with vehicle, DEC alone at 0.2 mg/kgsubcutaneous 3×/week, THU-DEC (THU 8 mg/kg with DEC 0.1 mg/kg)subcutaneous 3×/week or THU-DEC 3×/week alternating with THU-5AZA3×/week (THU 8 mg/kg with 5AZA 1 mg/kg). n=5 per treatment group. Asevident from FIG. 9 the alternating regimen of THU-Dec with Thu-5Azasignificantly increases the survival probability in these xenotransplantmodels of patient-derived AML.

What is claimed is:
 1. A method for treating a cancer or a blooddisorder in a subject, the method comprising: administering a firstcomposition to the subject comprising decitabine, tetrahydrouridine anda pharmaceutically acceptable carrier, administering a secondcomposition to the subject comprising 5-azacytidine, tetrahydrouridineand a pharmaceutically acceptable carrier, wherein the first and thesecond compositions are administered to the subject alternately.
 2. Themethod of claim 1 wherein the first composition administered to thesubject comprises about 1 to about 10 mg/m² of decitabine and about 400to 500 mg/m² of tetrahydrouridine.
 3. The method of claim 1, wherein thesecond composition administered to the subject comprises about 10 toabout 100 mg/m² of 5-azacytidine and about 400 to 500 mg/m² oftetrahydrouridine.
 4. The method of claim 1, wherein administration ofthe first and second compositions result in a peak decitabine plasmaconcentration of less than about 0.5 μM and a peak 5-azacytidine plasmaconcentration of not more than 5 μM in the subject.
 5. The method ofclaim 1, wherein the tetrahydrouridine in the first and secondcomposition is bio-available about 1 minute to about 180 minutes beforedecitabine or 5-azacytidine.
 6. The method of claim 1, wherein thetetrahydrouridine in the first and second compositions is a2′-fluorinated tetrahydrouridine derivative and is bio-available about15 minutes to about 180 minutes before decitabine or 5-azacytidine. 7.The method of claim 1, wherein the first composition is present in afirst pill and the second composition is present in a second pill. 8.The method of claim 1, wherein the cancer is a benign, metastatic orrefractory cancer of any tissue.
 9. The method of claim 1, wherein theblood disorder is sickle cell disease, thalassemia or hemoglobinopathy.10. The method of claim 1, wherein the first and the second compositionare administered after measuring a predictive biomarker in the subject.11. The method of claim 10, wherein the measurement of the predictivebiomarker includes the deoxycytidine kinase levels in the subject. 12.The method of claim 1, wherein the subject is a mammal.
 13. The methodof claim 1, wherein the 5-azacytidine and the tetrahydrouridine aredifferentially located in the second composition, and wherein thetetrahydrouridine is bio-available about 1 minute to about 180 minutesbefore the 5-azacytidine.
 14. The method of claim 1, wherein the secondcomposition comprises about 10 to about 100 mg/m² of 5-azacytidine andabout 400 to 500 mg/m² of tetrahydrouridine, and wherein thetetrahydrouridine is bio-available about 1 minute to about 180 minutesbefore the 5-azacytidine.
 15. The method of claim 1, wherein the secondcomposition comprises about 10 to about 100 mg/m² of 5-azacytidine andabout 400 to 500 mg/m² of tetrahydrouridine, wherein the secondcomposition is in form of a pill or tablet or capsule, wherein thetetrahydrouridine is located on the surface and the 5-azacytidine islocated inside the pill or tablet or capsule, and wherein thetetrahydrouridine is bio-available about 1 minute to about 180 minutesbefore the 5-azacytidine.